Protein-adsorbing material and method for producing the same

ABSTRACT

An object of the invention is to provide a protein-adsorbing material having both adsorption capacity and high-speed treatment performance applicable to not only analysis use but also industrial use, in balance, and to provide a method for producing the same. The present invention provides a protein-adsorbing material comprising a polymer base-material, a polymer side-chain immobilized to the surface of the polymer base-material and a functional group having protein adsorption ability and immobilized to the polymer side-chain, in which the mass of the polymer side-chain is 5 to 30% relative to the mass of the polymer base-material.

TECHNICAL FIELD

The present invention relates to a protein-adsorbing material and amethod for producing the same.

BACKGROUND ART

Conventionally, a purification operation by adsorption, such asrecovering a valuable substance such as a protein by adsorption orremoving impurities by adsorption, performed in a bioprocess, such as apharmaceutical product production process, has been performed by passinga solution to be treated through a column charged with porous gel beadshaving a particle size beyond 100 μm and serving as an adsorbent. As thegel beads, beads formed of a polysaccharide such as cellulose, dextranand agarose are frequently used. These beads which are porous beads,have numeral micropores within a bead particle, and the beads acquire acapacity of adsorbing a desired substance by increasing a specificsurface area by providing micropores. A crude-material solution, whichcontains a desired substance and impurities and obtained by e.g.,cultivating, is passed through the column charged with the porous gelbeads and the desired substance or impurities are separated by beingadsorbed by a functional group having protein adsorption ability andimmobilized to the micro pore surface when the solution passes throughthe micropores. However, conventional gel beads have a problem. A largeresistance is offered to the migration of a substance into a gel beadparticle, more specifically, to the diffusion of a substance through themicropores. Therefore, as the loading speed of a crude-material solutionto a column increases, a functional group within micropores is not usedfor adsorption and only the functional group present on the outersurface of the gel bead particles are used for adsorption. As a result,an adsorption capacity greatly decreases. Purification by adsorption isnot easily performed at a high speed.

On the other hand, gel beads formed of nonporous particles, in whichonly a functional group immobilized to the outer surface of theparticles functions, have an advantage in that even if a loading speedof a liquid increases, a decrease of adsorption capacity is low.However, since the absolute value of the specific surface area of suchgel beads is small, the adsorption capacity sufficient for industrialuse is not easily obtained, and use of the gel beads mostly remains foranalysis.

Furthermore, studies have been conducted on a method, in which afunctional group is immobilized to a micropore surface of a porousmembrane such as a micro filter and a solution to be treated is forciblypassed through micropores by filtration (see, for example, Non-PatentDocuments 1 and 2). According to the method, even if a solution isloaded at a high speed, the functional group within a micropore can beefficiently used. Thus, a decrease of adsorption capacity rarely occurs.

Non-Patent Document 1: Kyoichi Saito et al., “Chemical Engineering”,August issue, 1996, pp. 25-28

Non-Patent Document 2: Noboru Kubota, “Radiation and Industry”,December, 1998, No. 80, pp. 45-47

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

However, when a porous membrane is used like in the methods described inNon-Patent Documents 1 and 2, a solution is passed by filtration, thethickness of the porous membrane has to be reduced to some extent suchthat filtration pressure may not be increased excessively high. If so,it is difficult to increase the adsorption capacity (absolute value) inthe membrane-thickness direction.

It is an object of the present invention to provide a protein-adsorbingmaterial having both an adsorption capacity and high-speed treatmentperformance applicable to not only analysis use but also industrial usein balance and providing a method of producing the same.

Means for Solving the Problem

The present inventors have conducted intensive studies with a view tosolving the aforementioned problem, and, as a result, have found thatboth a high speed adsorption treatment and a high adsorption capacitycan be attained in balance by immobilizing a polymer side-chain to whicha functional group having protein adsorption ability is immobilized, tothe surface of a polymer base-material, which may not be porous, at apredetermined ratio, and that a protein-adsorbing material having both ahigh-speed adsorption treatment and a high adsorption capacity inbalance can be produced by use of a radiation graft polymerizationmethod. As a result that the present inventors have further conductedintensive studies, they found that the amount of polymer side-chains tobe formed on the surface of a base material can be maximized at anextremely small grafting ratio that may never ever been conceived, andhave accomplished the present invention.

More specifically, the present invention is as follows.

(1) A protein-adsorbing material comprising a polymer base-material, apolymer side-chain immobilized to the surface of the polymerbase-material and a functional group having protein adsorption abilityand immobilized to the polymer side-chain, in which the mass of thepolymer side-chain is 5 to 30% relative to the mass of the polymerbase-material.

(2) The protein-adsorbing material according to above item (1) in whichthe polymer side-chain is obtained by polymerization of a vinyl monomer.

(3) The protein-adsorbing material according to above item (1) or (2),in which the polymer side-chain is immobilized to the surface of thepolymer base-material by binding to a polymer constituting the polymerbase-material and the functional group is immobilized by binding to thepolymer side-chain.

(4) A method for producing a protein-adsorbing material comprising afirst step of activating a polymer base-material; a second step ofbringing, with the polymer base-material activated, a vinyl monomerhaving a functional group having protein adsorption ability or a vinylmonomer having a functional group, to which a functional group havingprotein adsorption ability can be introduced, into contact, therebyimmobilizing a polymer side-chain formed by polymerization of the vinylmonomer to the surface of the polymer base-material; and a third step ofintroducing a functional group having protein adsorption ability intothe functional group, to which the functional group having proteinadsorption ability can be introduced, and which are present in the vinylmonomer, when the vinyl monomer does not have the functional grouphaving protein adsorption ability, in which the mass of the polymerside-chain is 5 to 30% relative to the mass of the polymerbase-material.

(5) The method for producing a protein-adsorbing material according tothe above item (4), in which a polymer compound constituting the polymerbase-material comprises polyethylene, and, in the first step, thepolymer base-material is exposed to radiation with an irradiation doseof 1 to 20 kGy to activate the polymer base-material.

(6) The method for producing a protein-adsorbing material according tothe above item (5), in which, in the second step, the polymer side-chainis immobilized in a solution of the vinyl monomer of 30° C. or less.

(7) The method for producing a protein-adsorbing material according tothe above item (5) or (6), in which, in the second step, the vinylmonomer is brought into contact with the polymer base-materialactivated, in a solution prepared such that the concentration of thevinyl monomer is controlled to be not more than 10% by volume.

(8) The method for producing a protein-adsorbing material according toany one of the above items (5) to (7), in which the vinyl monomercomprises glycidyl methacrylate.

Advantage of the Invention

The present invention can provide a protein-adsorbing material havingboth an adsorption capacity and high-speed treatment performanceapplicable to not only analysis use but also industrial use in balanceand provide a method for producing the same.

BEST MODE FOR CARRYING OUT OF THE INVENTION

The best mode for carrying out of the invention (hereinafter, referredto as “the embodiment”) will be more specifically described below. Notethat, the present invention is not limited to the following embodimentand may be carried out by modifying it within the gist of theembodiment.

The protein-adsorbing material according to the embodiment contains apolymer compound as a base material. In the specification, the basematerial is also described as a “polymer base-material”.

The polymer compounds may include, for example, polyolefin such aspolyethylene and polypropylene, halogenated polyolefin such aspolyfluorinated vinylidene, a copolymer of an olefin and a halogenatedolefin and a mixture of these. Of them, it is preferred that the polymercompound contains polyethylene. Polyethylene is available at low costand excellent in chemical resistance and processability, and further,low in hygroscopic property and water absorptivity. In addition to this,polyethylene is rarely collapsed by radiation and relatively abundantlyhas crystal portions, which hold radicals from which a graftpolymerization reaction triggered by radiation exposure is initiated.Therefore, polyethylene is suitable for radiation graft polymerization.The content of polyethylene in the polymer compound is preferably 50 to100% by mass.

Polyethylene is roughly classified into low-density polyethylene andhigh-density polyethylene and both of them can be used in theembodiment. In view of stability of a polymer base-material in variousenvironments used in practice, high-density polyethylene having a highdegree of crystallization is relatively preferred.

Polyethylene may be a homopolymer of ethylene or may be a polymer towhich propylene and butene are added in order to control linearity anddensity.

The larger the molecular weight of polyethylene, the more preferable inview of stability of a polymer base-material in various environmentsused in practice. Particularly, a super-high molecular weight type ofpolyethylene, which has a weight average molecular weight of 1 millionor more, is preferred since mechanical properties thereof are alsoexcellent. The weight average molecular weight used herein is obtainedbased on measurement by gel permeation chromatography using polystyreneas a reference material.

The form of a polymer base-material is not particularly limited and maybe any one of the forms such as particle, non-woven cloth, woven clothand filament form.

A particulate polymer base-material is preferable since it can be usedas a filler to a conventional column. The particle may havetrue-spherical form or indeterminate form. Furthermore, a base materialmay be formed of primary particles, secondary particles, which areformed by coagulation of a plurality of primary particles into one body,or pulverized secondary particles.

A particle size of the particulate polymer base-material is preferably10 to 80 μm in average. The average particle size is expressed by thearithmetic mean of sizes of not less than 50 particles. The particlesize is expressed by an arithmetic mean, which is obtained by measuringthe minor axis and the major axis of each of the particles in amagnified photograph thereof. When the average particle size is largerthan 80 μm, the specific surface area decreases, with the result thatthe amount of functional group that can be introduced into a basematerial decreases and the adsorption capacity of a protein tends todecrease. When the average particle size is less than 10 μm, the spacebetween particles decreases, with the result that pressure lossincreases when a crude-material solution is passed through a columncharged with a protein-adsorbing material, and consequently, excessivepressure tends to be required when a crude-material solution is suppliedin practice. The particle size is preferably 10 to 40 μm in average.

The polymer base-material is preferably porous; however, a nonporouspolymer base-material can be used. To use the nonporous polymerbase-material as a protein-adsorbing material, a protein-adsorbingmaterial attaining both the high-speed adsorption treatment and highadsorption capacity in balance must be realized and adsorption of aprotein must be performed with a maximum efficiency. Then, how to designimmobilization of a functional group having protein adsorption abilityto a base material is the most important.

What is first required for immobilizing the functional group to apolymer base-material is immobilizing a polymer side-chain, via whichthe functional group is immobilized, to the base material. The polymerside-chain is immobilized to the base material, for example, by graftpolymerization. By virtue of immobilization of a functional group inthis manner, a functional group can be arranged not only on abase-material surface itself but also in the above space apart from thebase-material surface via the polymer side-chain immobilized to thebase-material surface. In a general method for immobilizing a functionalgroup to a base material, the functional group is immobilized only tothe base-material surface itself, that is, the two-dimensional surface.In contrast, in the method according to the embodiment, a functionalgroup is immobilized via the polymer side-chain immobilized to a basematerial. By virtue of this structure, a three-dimensional space abovethe base material, in which a polymer side-chain is extended, can beused as an immobilization space for the functional group. Therefore, themethod of the embodiment is overwhelmingly advantageous over theconventional method as mentioned above, in view of acquiring anadsorption capacity. The conventional method for immobilizing afunctional group to a base-material surface with no polymer side-chaininterposed between them has a so-called “adsorption space of aone-storied house type (plane surface type)”; whereas, the methodaccording to the embodiment for immobilizing a functional group to abase material with a polymer side-chain immobilized thereto interposedbetween them has a so-called “adsorption space of a many-storied housetype”.

Note that, in the specification, immobilization of a polymer side-chainto a polymer base-material and immobilization of a functional group to apolymer side-chain respectively mean binding of the polymer side-chainto a compound constituting the polymer base-material and binding of thefunctional group to the polymer side-chain. The binding may be made bycovalent bonding.

In the specification, the “polymer side-chain” refers to a polymer groupcapable of binding to a polymer compound constituting a polymerbase-material and serving as a main chain, as a side chain. It ispreferred that the polymer side-chain does not have many crosslinkedstructures. Since the molecule of a protein is generally large, it isdifficult for a protein molecule to enter into a crosslinked structureor move within the crosslinked structure, for example, when a polymerside-chain has a crosslinked structure such as styrene-divinyl benzene.As a result, only a part of the polymer side-chain is involved inadsorption, and virtually, it is difficult to adsorb a protein. Thepolymer side-chain preferably has a highly flexible methylene chain as amain chain thereof. The polymer side-chain having a methylene chain asthe main chain can be obtained, for example, by graft polymerization ofa vinyl monomer to a base material. Particularly preferable polymerside-chain is a polyglycidyl methacrylate chain. This polymer side-chaincan be formed by polymerization of glycidyl methacrylate, which is avinyl monomer. Glycidyl methacrylate polymerized has a highly reactiveglycidyl group. This is advantageous since various functional groups canbe introduced into an epoxy ring present in the glycidyl group by aring-opening addition reaction.

What is important to adsorb a protein with a maximum efficiency byimmobilizing a functional group having protein adsorption ability to apolymer base-material via a polymer side-chain and additionallyrealizing further higher speed adsorption treatment and higheradsorption capacity in balance, is how to set the grafting ratio of apolymer side-chain and how to set the density of a polymer side-chain.The grafting ratio of a polymer side-chain (unit: %) is calculated bythe expression:(mass of polymer side-chain (unit: g))/(mass of polymer base-materialbefore graft polymerization (unit: g))×100.

The density of a polymer side-chain is calculated by the expression:(mass of polymer side-chain (unit: g))/(surface area of polymerbase-material before graft polymerization (unit: m²)).

The mass (unit: g) of a polymer side-chain is calculated by theexpression:(total mass (unit: g) of polymer base-material after graftpolymerization and polymer side-chain)−(mass (unit: g) of polymerbase-material before graft polymerization).

Generally, it is considered beneficial that a grafting ratio isincreased to introduce a large number of functional groups, in order toattain the object of the invention. However, it is unexpectedly foundthat if the grafting ratio is excessively large, both the adsorptioncapacity and elution ratio of a protein-adsorbing material decrease.From this, it was found that there is a more preferable range of properdensity of a polymer side-chain. The reason for these is notspecifically elucidated; however, the present inventors presume thefollowing one as one of the causes. That is, the polymer side-chainsimmobilized to a polymer base-material may conceivably present like“whiskers”, which is immobilized on the surface of the base material.When the grafting ratio exceeds a predetermined level, it becomesdifficult for a protein to enter into a layer of a polymer side-chain,and a three-dimensional many-storied adsorption space formed of thepolymer side-chain layer is rarely used efficiently. As a result, aneffective adsorption capacity may decrease. Furthermore, it becomesdifficult for a part of proteins “entering into” the layer of a densepolymer side-chain to “creep out” of the polymer side-chain layer duringelution treatment. An elution ratio or a recovery rate may possiblydecrease.

Note that, the “elution ratio” refers to a ratio (%) of protein elutedfrom a protein-adsorbing material by an eluate based on the proteinadsorbed to the protein-adsorbing material, and corresponds to arecovery rate of the protein adsorbed to a protein-adsorbing material.

Although the preferable range of the grafting ratio and density of apolymer side-chain according to the embodiment is significantly narrowcompared to the preferable range so far presumed, the adsorptioncapacity is resulted in an increase. This is an extremely unexpectedfinding, which may not be obtained by extension of a conventional way ofthinking.

The grafting ratio of a polymer side-chain immobilized to a polymerbase-material falls within the range of 5 to 30%, and more preferablyfalls within the range of 10 to 20%. When the grafting ratio is lessthan 5%, a sufficient protein adsorption capacity cannot be obtained.When the grafting ratio is larger than 30%, an elution ratio decreases.Both cases are not preferable.

Furthermore, the density of a polymer side-chain immobilized to apolymer base-material is preferably 0.1 g/m² or more and less than 3g/m², more preferably 0.2 to 1.5 g/m², and further preferably 0.3 g/m²or more and less than 1.0 g/m². In the range of the density less than0.1 g/m², the adsorption capacity of a protein tends not to besufficiently obtained. When the density is 3 g /m² or more, the elutionratio tends to decrease. Both cases are not preferable.

In either case, in the embodiment, compared to a conventional graftingtechnique for imparting a function, a grafting ratio is low and thus theamount of functional group to be introduced into a protein-adsorbingmaterial is low. Nevertheless, the protein adsorption ability isimproved. This should be said to be an unexpected result.

The protein-adsorbing material of the embodiment contains a functionalgroup having protein adsorption ability. The “protein adsorptionability” used herein refers to an ability to adsorb a protein withoutmodifying the molecule of the protein. A functional group having proteinadsorption ability is roughly classified into four groups: (1) ionexchange adsorption type, (2) hydrophobic interaction adsorption type,(3) group-specific affinity adsorption type and (4) individual-specificaffinity adsorption type. Specific examples are as follows.

The functional groups of the ion exchange adsorption type may include,for example, a cation group such as a sulfonic acid group, a carboxylicacid group and a phosphoric acid group; an anionic group such as aquaternary ammonium salt group, a pyridinium salt group and tertiary tosecondary amino groups; and a chelate group such as an iminodiaceticacid group, a mercapto group and an ethylenediamine group.

The functional groups of the hydrophobic interaction adsorption type mayinclude, for example, a phenyl group and an alkyl group.

The functional group of the group-specific affinity adsorption type mayinclude, for example, a Cibacron Blue F3G-A, Protein A, concanavalin A,heparin, tannin and a metal chelate group.

The functional group of the individual-specific affinity adsorption typemay include, for example, antigens and antibodies.

To a polymer side-chain immobilized to a polymer base-material, thesefunctional groups having protein adsorption ability may be immobilizedsingly or in combination with two types or more. Furthermore, to thepolymer side-chain, not only a functional group having proteinadsorption ability but also a hydroxy group is desirably immobilized inorder to suppress non-specific adsorption and non-reversible adsorptionof a protein.

The protein-adsorbing material according to the embodiment, a functionalgroup having protein adsorption ability is preferably contained in anamount of not less than 0.1 mmol/g per mass (dry mass) of theprotein-adsorbing material. As described above, as a grafting ratiodecreases, the amount of functional group decreases. However, if thegrafting ratio of polymer side-chain immobilized to a polymerbase-material falls within the above range, the larger the amount offunctional group having protein adsorption ability to be immobilized toa polymer side-chain, the more preferable, since adsorption abilityincreases. The upper limit of the amount of functional group issubstantially 0.5 mmol/g.

The protein-adsorbing material according to the embodiment is producedby, for example, a radiation graft polymerization method using a polymercompound as a base material. More specifically, the method for producingthe protein-adsorbing material according to the embodiment contains afirst step of activating a polymer base-material and a second step ofbringing, with the polymer base-material activated, a vinyl monomerhaving a functional group having protein adsorption ability or a vinylmonomer having a functional group, to which a functional group havingprotein adsorption ability can be introduced, into contact, therebyimmobilizing a polymer side-chain formed by polymerization of a vinylmonomer to the surface of the polymer base-material. The method forproducing a protein-adsorbing material according to the embodiment maycontain a third step of introducing a functional group having proteinadsorption ability into the functional group (present in the vinylmonomer), to which the functional group having protein adsorptionability can be introduced, when the vinyl monomer does not have thefunctional group having protein adsorption ability.

The vinyl monomers having a functional group having protein adsorptionability and used in the second step may include, for example, sodiumstyrene sulfonate having a sulfonic acid group as a functional group;and an acrylic acid having a carboxyl group as a functional group.Furthermore, as the functional group to which a functional group havingprotein adsorption ability can be introduced, a highly reactivefunctional group is preferable and the functional groups may include,for example, an epoxy ring, a hydroxy group and an amino group. Of them,an epoxy ring, since it is reactive with a wide variety of molecules, isparticularly used effectively as the functional group, to which afunctional group having protein adsorption ability can be introduced.The vinyl monomers having a functional group, to which a functionalgroup having protein adsorption ability can be introduced, may includeglycidyl methacrylate having an epoxy ring as a functional group andvinyl acetate having an acetic acid ester residue capable of forming ahydroxy group by hydrolysis as a functional group. These may be usedsingly or as a mixture of two or more types. The vinyl monomerpreferably contains glycidyl methacrylate. The amount of glycidylmethacrylate relative to the total amount of vinyl monomer is preferably50 to 100% by mass.

Furthermore, as a method for introducing various functional groupshaving protein adsorption ability into an epoxy ring in the third step,the method described in e.g., Non-Patent Document 2 as mentioned abovemay be employed.

The polymer side-chain formed by polymerizing a vinyl monomer is apolymer side-chain (hereinafter referred to as a “graft polymer chain”)formed by polymerizing a vinyl monomer in accordance with graftpolymerization. Immobilization to a polymer base-material is initiatedby activating the surface of the base material such that a vinyl monomercan be polymerized (in the first step). Methods for activating thebase-material surface may include a method for generating radicals onthe base-material surface. By virtue of this, graft polymerization of avinyl monomer can be started using the generated radicals as a startingpoint. As a method for generating radicals on a base-material surface,radiation exposure for generating radicals is particularly preferable inview of generating radicals uniformly over the entire surface of thebase material. A polymer chain may be produced by graft polymerizationusing radicals as a starting point. The radiation preferably used in theembodiment is ionizing radiation such as α, β and γ beams and electronbeam. Of these all of which can be used, particularly, an electron beamor a γ beam is suitably used.

Note that, to immobilize a graft polymer side-chain with an appropriatedensity to a polymer base-material, it is important to set the amount ofradicals to be generated, which serves as a starting point for graftpolymerization, to fall within an appropriate range. Specifically, whena polymer base-material is activated by radiation exposure in the firststep, the irradiation dose (of radiation) to a base material isimportant. In the embodiment, it is a key point that the irradiationdose (of radiation) is lower than conventional one. In particular, toefficiently produce a necessary amount of radicals when a polymercompound constituting a polymer base-material contains polyethylene, theirradiation dose is preferably 1 to 20 kGy, and further preferably 1 to10 kGy. As previously described, in the embodiment, the density ofpolymer side-chains to be immobilized to the polymer base-materialsurface is important. The amount of radicals serving as an origin ofgrafting a polymer side-chain is determined by the irradiation dose.Therefore, if the irradiation dose is controlled to be within the aboverange, the structure of a polymer side-chain may conceivably beoptimized.

Methods for grafting a polymer to a polymer base-material by use ofradiation (hereinafter sometimes referred to as a “radiation graftpolymerization method”) may include, for example, a pre-irradiationmethod, in which a polymer base-material is previously exposed toradiation, and thereafter, brought into contact with a vinyl monomer ata radical serving as an origin; and a simultaneous radiation method, inwhich a vinyl monomer solution is exposed to radiation. Of these, themethod capable of providing stable graft polymerization is thepre-irradiation method.

Methods for graft polymerization in accordance with radicalpolymerization by bringing a vinyl monomer into contact with radicalsgenerated in a polymer base-material may include a gas-phase method, inwhich a vinyl monomer vaporized in a gas phase is brought into contact,and a liquid-phase method, in which a liquid-state vinyl monomer isdirectly brought into contact or a liquid-state vinyl monomer is dilutedwith a solvent and brought into contact in the solution. By the reasonthat a graft amount, in other words, the density of the polymerside-chain grafted to a base material, is easily controlled, theliquid-phase method, in which a vinyl monomer is diluted with a solventand brought into contact in the solution, is preferable. As the solvent,a solvent having a small swelling property to a resin (polymer compound)constituting a base material is preferably used in order to limit agraft polymerization reaction to occur near the surface of the basematerial while suppressing the reaction from occurring in the innermostpart of the base material (for example, in the case of using aparticular base, in the innermost part of a particle). Specifically, asolvent having a degree of swelling the resin (constituting a basematerial) of 10% or less, is preferable. When the resin constituting abase material is polyethylene, for example, an alcohol such as methanol,ethanol, isopropyl alcohol and butanol etc., is preferably used as thesolvent. The “degree of swelling” is expressed by a percentage value ofthe difference between “the diameter of a resin particle soaked in asolvent for one hour” at room temperature and “the diameter of a resinparticle before soaking” divided by “the diameter of a resin particlebefore soaking”.

When an alcohol is used as the solvent and glycidyl methacrylate isemployed as the vinyl monomer, it is preferred that a polymer side-chainis immobilized in a vinyl monomer solution of 30° C. or less in order toconduct a grafting reaction between a polymer compound constituting apolymer base-material and glycidyl methacrylate in an alcohol solvent.At this time, when the temperature of the vinyl monomer solution exceeds30° C., the reaction rate increases, with the result that it becomesdifficult to control the reaction so as to have a predetermined graftingratio. The temperature of the vinyl monomer solution is more preferably0 to 20° C.

The concentration of a vinyl monomer in the vinyl monomer solution ispreferably not more than 10% by volume. More specifically, theconcentration of glycidyl methacrylate in an alcohol solvent ispreferably not more than 10% by volume. When the concentration exceeds10% by volume, it tends to be difficult to obtain a protein-adsorbingmaterial having a large adsorption capacity.

The temperature of a reaction mixture and the concentration of glycidylmethacrylate conceivably have a large effect upon the density of apolymer side-chain to be formed on a polymer base-material surface. Morespecifically, it is considered that they may be important factors forforming a structure which allows protein molecules to easily enter intothe space between polymer side-chains.

As a specific example of a particularly preferable method for producinga protein-adsorbing material according to the embodiment, a radiationgraft polymerization method is mentioned using a polyethylene particleas a polymer base-material and glycidyl methacrylate as a vinyl monomer.The average size of the polyethylene particle is preferably 10 to 80 μm,more preferably, 10 to 60 μm, and further preferably 10 to 40 μm. As theradiation graft polymerization method, a pre-irradiation method ispreferred. The graft polymerization of glycidyl methacrylate to a basematerial having radicals generated thereon is preferably performed in analcohol solution of glycidyl methacrylate. As the alcohol, methanol,ethanol, isopropyl alcohol and butanol can be suitably used. Asdescribed above, an alcohol, which is a reaction solvent having a smallswelling property to a resin constituting a base material, is preferablyused in order to limit a graft polymerization reaction to occur near thesurface of the base material while suppressing the reaction fromoccurring in the innermost part of particles of the base material. Analcohol has a small degree of swelling to polyethylene.

It is possible to control a grafting ratio by controlling the reactiontemperature of the reaction solution, the concentration and reactiontime of glycidyl methacrylate.

The grafting ratio of glycidyl methacrylate is preferably 5 to 30%, andmore preferably 10 to 20%. It is possible to control the density ofpolymer side-chain to be immobilized to a polymer base-material within apreferably range by controlling the grafting ratio.

After glycidyl methacrylate is immobilized to a polymer base-material bygraft polymerization, a functional group having protein adsorptionability may be introduced into a polymer side-chain, i.e., polyglycidylmethacrylate side chain. To do this, a functional group may beintroduced to an epoxy ring of a glycidyl group in the polymerside-chain by a ring-opening addition reaction. For example, when acationic exchange group is introduced as a functional group havingprotein adsorption ability, a sulphite can be added to a glycidyl groupin the polymer side-chain immobilized by graft polymerization. Morespecifically, a method for introducing a sulfone group by reacting abase material, to which glycidyl methacrylate is immobilized by graftpolymerization, and sodium sulphite in a solution mixture ofwater/isopropyl alcohol can be employed. Furthermore, for example, whenan anionic exchange group is introduced as the functional group havingprotein adsorption ability, trimethylamine hydrochloride is reacted witha glycidyl group in the polymer side-chain immobilized by graftpolymerization. In this way, a quaternary ammonium group can beintroduced.

Note that, the surface area of a polymer base-material can be determinedby a mercury press-in method. Furthermore, a protein-adsorbing materialmay be protein-adsorbing beads (in the form of beads).

EXAMPLES

The embodiment will be more specifically described by way of examplesbelow; however, the embodiment is not be limited only to these examples.

Example 1

As a polymer base-material, super-high molecular weight polyethyleneparticles (GUR-2126, specific surface area: 0.18 m²/g, manufactured byTicona)—whose mass was previously measured—having an average particlesize of 35 μm, were prepared. The polyethylene particles were exposed toan electron beam of 10 kGy to generate radicals.

After radicals were generated, the polyethylene particles were soaked ina 2% by volume glycidyl methacrylate/1-butanol solution and shaken at 5°C. for 150 hours to conduct a graft polymerization reaction. Theparticles obtained were washed with alcohol and dried, and then, masswas measured. The grafting ratio, as calculated from the mass, was 17%.The density of a polymer side-chain was 0.9 g/m².

The particles obtained were soaked in a solution of sodium sulfite:isopropanol: pure water=10:15:75 (% by mass) and shaken at 80° C. for 12hours to introduce a sulfonic acid group serving as a functional grouphaving protein adsorption ability into a glycidyl group. The particleshaving a sulfonic acid group introduced therein were dried and measuredfor mass. Based on the mass increased, the amount of sulfonic acid groupimmobilized was obtained. The amount of sulfonic acid group immobilizedwas 0.3 mmol/g. Furthermore, the particles having a sulfonic acid groupintroduced therein were soaked in a 0.5 mol/L aqueous sulfuric acidsolution, and shaken at 80° C. for 2 hours to convert an unreactedglycidyl group to a diol. In this manner, a cationic protein-adsorbingbeads serving as a protein-adsorbing material were obtained.

The protein-adsorbing beads obtained were loaded in a column having asectional area of 0.39 cm² (height of charged beads: 3 cm), and testedfor the following adsorption performance. First, a 2 g/L lysozymesolution (a 10 mmol aqueous solution of sodium carbonate/sodiumhydroxide as a buffer solution, pH=9) was passed as a protein solutionserving as a crude-material solution through the column from the top tothe bottom at a space velocity of 200 h⁻¹. In this manner, an operationof adsorbing lysozyme was performed. A discharge solution from a liquidoutlet port under the column was sampled, and the concentration oflysozyme in the discharge solution was monitored by absorptiometry(absorption wavelength: 280 nm). The concentration of lysozyme in thedischarge solution was zero at the beginning; however, as the amount ofsolution to be loaded was increased, lysozyme gradually leaked out andthe lysozyme concentration increased. An adsorption operation wascontinued until the concentration of lysozyme in the discharge solutionreached the same concentration (2 g/L) as the original solution. Theadsorption amount until the concentration of lysozyme in the dischargesolution reached 1/10 that of the original solution was determined as adynamic adsorption capacity. The adsorption amount until theconcentration of lysozyme in the discharge solution reached the same asthat of the original solution was determined an equivalent adsorptioncapacity. After completion of the adsorption operation, the buffersolution was passed through the column to wash it. Thereafter, a 0.5mol/L aqueous sodium chloride solution was passed through the column asan eluate to elute lysozyme adsorbed to the protein-adsorbing material.An elution ratio was calculated in accordance with an expression:100×(elution amount)/(equivalent adsorption amount (the same unit wasused in the elution amount)). As a result, the dynamic adsorptioncapacity was 39 mg/mL (per column charge volume, the same shall applyhereinafter). The equivalent adsorption capacity was 60 mg/mL and theelution ratio was 100%.

Comparative Example 1

A comparative example where the irradiation dose was large and thegrafting ratio was large, was performed as follows. Cationicprotein-adsorbing beads were obtained in the same manner as in Example 1except that the irradiation dose of the electron beam was changed from10 kGy to 100 kGy and the time of the graft polymerization reaction waschanged from 150 hours to 24 hours. The grafting ratio of the beads was45% and the density of polymer side-chain was 2.5 g/m². The amount ofsulfonic acid group immobilized was 1.2 mmol/g.

The protein-adsorbing beads obtained were tested for adsorptionperformance in the same manner as in Example 1. The dynamic adsorptioncapacity was 14 mg/mL, the equivalent adsorption capacity was 22 mg/mL,and the elution ratio was 90%. As a result, the adsorption capacity didnot reach a half of that obtained in Example 1.

Comparative Example 2

A comparative example where reaction temperature was high and thegrafting ratio was large was performed as follows. Cationicprotein-adsorbing beads were obtained in the same manner as in Example 1except that the reaction temperature of the graft polymerization waschanged from 5° C. to 40° C. and the reaction time was changed from 150hours to 2 hours. The grafting ratio of the beads was 41% and thedensity of polymer side-chain was 2.3 g/m². The amount of sulfonic acidgroup immobilized was 1.1 mmol/g.

The protein-adsorbing beads obtained were tested for adsorptionperformance in the same manner as in Example 1. The dynamic adsorptioncapacity was 20 mg/mL and the equivalent adsorption capacity was 31mg/mL. They were about a half of those obtained in Example 1.Furthermore, the elution ratio was 85%. It was difficult to sufficientlyrecover the protein adsorbed.

Example 2

As a polymer base-material, super-high molecular weight polyethyleneparticles (GUR-2126, specific surface area: 0.18 m²/g, manufactured byTicona)—whose mass was previously measured—having an average particlesize of 35 μm were prepared. The polyethylene particles were exposed toan electron beam of 10 kGy to generate radicals.

After radicals were generated, the polyethylene particles were soaked ina 2% by volume glycidyl methacrylate/1-butanol solution and shaken at 5°C. for 120 hours to perform a graft polymerization reaction. Theparticles obtained were washed with alcohol and dried, and mass wasmeasured. The grafting ratio, as calculated from the mass, was 13%. Thedensity of a polymer side-chain was 0.7 g/m².

The particles obtained were soaked in a 50% by volume aqueousdiethylamine solution and shaken at 30° C. for 24 hours to introduce adiethyl amino group serving as a functional group having proteinadsorption ability into a glycidyl group. The particles having a diethylamino group introduced therein were dried and measured for mass. Basedon the mass increased, the amount of diethyl amino group immobilized wasobtained. The amount of diethyl amino group immobilized was 0.6 mmol/g.Next, the particles having a diethyl amino group introduced therein weresoaked in a 50% by volume ethanolamine/methanol solution, and soaked at30° C. for 24 hours to etanolaminate an unreacted glycidyl group. Inthis manner, an anionic protein-adsorbing beads serving as aprotein-adsorbing material were obtained.

The protein-adsorbing beads obtained were loaded in the same column asused in Example 1 (height of charged beads: 3 cm), and tested for thefollowing adsorption performance. First, a 1 g/L bovine serum albuminsolution (20 mmol Tris-HCl buffer, pH=8) was passed as a proteinsolution serving as a crude-material solution through the column fromthe top to the bottom at a space velocity of 200 h⁻¹. In this manner, anoperation of adsorbing albumin was performed. A discharge solution froma liquid outlet port under the column was sampled, and the concentrationof albumin in the discharge solution was monitored by absorptiometry(absorption wavelength: 280 nm). The concentration of albumin in thedischarge solution was zero at the beginning; however, as the amount ofsolution to be loaded was increased, albumin gradually leaked out andthe albumin concentration increased. An adsorption operation wascontinued until the concentration of albumin in the discharge solutionreached the same concentration (1 g/L) as the original solution. Theadsorption amount until the concentration of albumin in the dischargesolution reached 1/10 that of the original solution was determined as adynamic adsorption capacity. The adsorption amount until theconcentration of albumin in the discharge solution reached the same asthat of the original solution was determined an equivalent adsorptioncapacity. After completion of the adsorption operation, the buffersolution was passed through the column to wash it. Thereafter, a 1 mol/Laqueous sodium chloride solution was passed through the column as aneluate to elute albumin adsorbed to the protein-adsorbing material. Anelution ratio was calculated in accordance with an expression:100×(elution amount)/(equivalent adsorption amount (the same unit usedin the elution amount)). As a result, the dynamic adsorption capacitywas 37 mg/mL, the equivalent adsorption capacity was 43 mg/mL and theelution ratio was 100%.

Example 3

Anionic protein-adsorbing beads were obtained in the same manner as inExample 2 except that the concentration of glycidyl methacrylate in thereaction solution was changed from 2% by volume to 15% by volume. Thegrafting ratio was 20%, the density of the polymer side-chain was 1.2g/m², and the amount of diethyl amino group immobilized was 1.1 mmol/g.

As the protein-adsorbing beads obtained were tested for the adsorptionperformance in the same manner as in Example 2. The dynamic adsorptioncapacity was 18 mg/mL and the equivalent adsorption capacity was 66mg/mL. The dynamic adsorption capacity was low compared to theequivalent adsorption capacity. Furthermore, the elution ratio was 95%.

Example 4

As a polymer base-material, super-high molecular weight polyethyleneparticles (GUR-2126, specific surface area: 0.18 m²/g, manufactured byTicona)—whose mass was previously measured—having an average particlesize of 35 μm were prepared. The polyethylene particles were exposed toan electron beam of 1 kGy to generate radicals.

After radicals were generated, the polyethylene particles were soaked ina 10% by volume glycidyl methacrylate/1-butanol solution and shaken at30° C. for 2 hours to perform a graft polymerization reaction. Theparticles obtained were washed with alcohol and dried, and then, masswas measured. The grafting ratio, as calculated from the mass, was 21%.The density of a polymer side-chain was 1.2 g/m².

To the particles obtained, a sulfone acid group serving as a functionalgroup having protein adsorption ability was introduced in the samemanner as in Example 1. The amount of sulfonic acid group immobilizedwas 0.5 mmol/g.

Furthermore, unreacted glycidyl group was converted into a diol in thesame manner as in Example 1. In this manner, cationic protein-adsorbingbeads were obtained as a protein-adsorbing material.

The protein-adsorbing beads obtained were tested for adsorptionperformance in the same manner as in Example 1. The dynamic adsorptioncapacity was 38 mg/mL and the equivalent adsorption capacity was 55mg/mL. Furthermore, the elution ratio was 98%. The protein was almostcompletely recovered.

Example 5

As a polymer base-material, super-high molecular weight polyethyleneparticles (GUR-2126, specific surface area: 0.18 m²/g, manufactured byTicona)—whose mass was previously measured—having an average particlesize of 35 μm were prepared. The polyethylene particles were exposed toy beam of 10 kGy to generate radicals.

After radicals were generated, the polyethylene particles were soaked ina 4% by volume glycidyl methacrylate/1-butanol solution and shaken at 5°C. for 100 hours to perform a graft polymerization reaction. Theparticles obtained were washed with alcohol and dried, and then, masswas measured. The grafting ratio, as calculated from the mass, was 10%.The density of a polymer side-chain was 0.5 g/m².

The particles obtained were soaked in a solution of sodium sulfite:isopropanol: pure water=10:15:75 (% by mass) and shaken at 80° C. for 12hours to introduce a sulfonic acid group serving as a functional grouphaving protein adsorption ability into a glycidyl group. The particleshaving a sulfonic acid group introduced therein were dried and measuredfor mass. Based on the mass increased, the amount of sulfonic acid groupimmobilized was obtained. The amount of sulfonic acid group immobilizedwas 0.2 mmol/g. Furthermore, the particles having a sulfonic acid groupintroduced therein were soaked in a 0.5 mol/L aqueous sulfuric acidsolution and shaken at 80° C. for 2 hours to convert an unreactedglycidyl group to a diol. In this manner, a cationic protein-adsorbingbeads serving as a protein-adsorbing material were obtained.

The protein-adsorbing beads obtained were tested for adsorptionperformance in the same manner as in Example 1. The dynamic adsorptioncapacity was 28 mg/mL and the equivalent adsorption capacity was 41mg/mL. The elution ratio was 100%.

The present application was based on Japanese Patent Application(Application No. 2007-304826) filed on Nov. 26, 2007 and the contentthereof is incorporated by reference herein.

Industrial Applicability

According to the present invention, it is possible to provide aprotein-adsorbing material, which is suitably used in a purificationoperation by adsorption, such as recovering a valuable substance such asa protein by adsorption, and removing impurities by adsorption in abioprocess such as a pharmaceutical product production process, andwhich allows purification by adsorption at a high speed and with a highadsorption capacity. More specifically, the protein-adsorbing materialof the present invention reduces a limitation on substance migration inan adsorbing material and diffusion resistance into micropores, therebyadsorbing and purifying a large molecule such as a protein. Accordingly,it is possible to provide a protein-adsorbing material having both anadsorption capacity (the adsorption capacity is not an equivalentadsorption capacity but the capacity of adsorbing until adsorptionleakage cannot be ignored during adsorption treatment, that is, adynamic adsorption capacity) applicable for industrial use or analysisuse and high-speed treatment performance in balance, and to provide amethod for producing the same.

The invention claimed is:
 1. A method for producing a protein-adsorbingmaterial comprising activating a polymer base-material by exposing saidpolymer base-material to radiation with an irradiation dose of 1 to 20kGy; bringing, with said polymer base-material activated, a vinylmonomer having a functional group having protein adsorption ability or avinyl monomer having a functional group, to which a functional grouphaving protein adsorption ability can be introduced, into contact, in asolution of the vinyl monomer, at 20° C. or less and for a period of 100to 150 hours, thereby immobilizing a polymer side-chain formed bypolymerization of said vinyl monomer to the surface of said polymerbase-material and introducing a functional group having proteinadsorption ability into said functional group, to which the functionalgroup having protein adsorption ability can be introduced, and which ispresent in said vinyl monomer, when said vinyl monomer does not havesaid functional group having protein adsorption ability, wherein themass of said polymer side-chain is 5 to 30% relative to the mass of saidpolymer base-material.
 2. The method for producing a protein-adsorbingmaterial according to claim 1, wherein a polymer compound constitutingsaid polymer base-material comprises polyethylene.
 3. The method forproducing a protein-adsorbing material according to claim 2, wherein, insaid second step, said vinyl monomer is brought into contact with saidpolymer base-material activated, in a solution prepared such that theconcentration of said vinyl monomer is controlled to be not more than10% by volume.
 4. The method for producing a protein-adsorbing materialaccording to claim 2, wherein the vinyl monomer comprises glycidylmethacrylate.